DNA multiphoton absorption generates localized damage for studying repair dynamics in live cells

Abstract

Investigations into the spatiotemporal dynamics of DNA repair using live-cell imaging are aided by the ability to generate well defined regions of ultravioletlike photolesions in an optical microscope. We demonstrate that multiphoton excitation of DNA in live cells with visible femtosecond pulses produces thymine cyclopyrimidine dimers (CPDs), the primary ultraviolet DNA photoproduct. The CPDs are produced with a cubic to supercubic power dependence using pulses in the wavelength range from at least 400 to 525 nm. We show that the CPDs are confined in all three spatial dimensions, making multiphoton excitation of DNA with visible light an ideal technique for generating localized DNA photolesions in a wide variety of samples, from cultured cells to thicker tissues. We demonstrate the utility of this method by applying it to investigate the spatiotemporal recruitment of GFP-tagged topoisomerase I (TopI) to sites of localized DNA damage in polytene chromosomes within live cells of optically thick Drosophila salivary glands.

Link

doi: 10.1016/j.bpj.2011.09.031
Analysis code is on Github

Recommended citation: Daddysman MK and Fecko CJ (2011) “DNA multiphoton absorption generates localized damage for studying repair dynamics in live cells”, Biophys J, 101:9, 2294-2303.